DESCRIPTION: Bax, a member of the Bcl-2 family, has been shown to be inhibitory to Bcl-2, in most cases promoting cell death. For example, Bax levels are elevated in neurons in the area penumbra following focal ischemia and have also been seen to elevate in spinal motor neurons of ALS patients. Additionally, reductions in Bax expression achieved through antisense or gene knockout have been protective to "at risk" neurons. However, the mechanism by which Bax facilitates or allows apoptosis or necrosis remains unknown. The principal investigator's lab has identified, cloned and sequenced cDNAs for two Bax inhibitory proteins, BI-1 and BI-2, selected by screening a human cDNA library for the ability to suppress Bax-induced killing in yeast. These are integral membrane proteins found largely in the endoplasmic reticulum but perhaps in other internal cytoplasmic membranous systems (e.g., mitochondria), as well. BI-1 has topographical transmembrane folds similar to presenilin-1 and -2, while BI-2 has a RING domain that appears responsible for its suppression of Bax-induced apoptosis in mammalian cells. The principal investigator hypothesizes that BI-1 and -2 are physiologically relevant to control of apoptotic and perhaps certain necrotic pathways in the brain both during normal development and in disease states such as ischemia. To test this hypothesis, 5 groups of studies are proposed: 1. Transfection experiments of BI-1 and -2 into neuronal cell culture models of cell death; 2. Structure-function analysis of BI-1 and -2 mutants in said models; 3. Immunolocalization studies of BI-1 and -2 proteins in apoptotic cell models, neuronal cell models, and brain; 4. Expression studies of BI-1 and -2 in normal brain and following focal ischemia; and 5. Generations of BI-1 and -2 knockout and transgenic mice to provide an in vivo experimental context to the studies proposed in the first four aims. The overall goal of the proposal is to understand basic mechanisms contributing to neuronal death in hopes of preventing the processes following acute events such as ischemia and slowing or halting the process during diseases of chronic neurodegeneration.